PROJECT SUMMARY DNA methylation, an epigenetic factor, plays an important role in regulating gene expression and alterations in DNA methylation is a feature associated with a number of human diseases. Inflammation and environmental factors such as psychophysical stress induces demethylation of pro-nociceptive genes leading to their aberrant expression. The objective of this renewal application is to investigate how muscle inflammation remotely regulates DNA methylation of multiple pro-nociceptive genes in trigeminal ganglia (TG) that have been implicated in pain and hyperalgesia. Our central hypothesis is that masseter muscle inflammation results in reduced methylation of pro-nociceptive genes in TG leading to their aberrant expression, which contributes to the development of pain and mechanical hyperalgesia. We further hypothesize that psychophysical stress potentiates these effects via the excess production of reactive oxygen species (ROS) within TG, which regulate DNA methylation. In Aim 1, we will determine the role of DNA methylation in inflammatory pain responses. Specifically, we will examine whether increased DNA methylation via DNMTs at the promoter region of individual pro-nociceptive genes, prevents inflammatory pain and hyperalgesia. We have confirmed that pro-nociceptive genes such as TRPV1, TRPA1, P2X3 and PIEZO2 contain CG islands that bind DNMTs and that the inhibition of DNMT activities increased their expression in TG. In order to determine the role of DNA methylation in individual genes, we designed and validated a novel DNMT fusion protein complex that targets the promoter region of a specific gene, using the CRISPR-dCAS9 technology. We expect that the expression of the fusion protein within TG will prevent the upregulation of the target gene and reveal the relative contribution of DNA methylation for a specific gene in pain and hyperalgesia under a myositis condition. In Aim 2, we will investigate the role of intraganglionic ROS in DNA methylation of pro-nociceptive genes. We will examine whether ROS regulates DNA methylation of TRPV1 and TRPA1 genes in TG and whether stress elevates intraganglionic ROS, which maintains the reduced level of methylation of the pro-nociceptive genes. Our preliminary data suggest ROS as a key upstream factor involved in DNA methylation of the two pro-nociceptive genes. We predict that the blockade of ROS accumulation in TG or targeted methylation of DNA promoters will prevent stress-mediated potentiation of hyperalgesia and the upregulation of TRPV1 and TRPA1. Successful achievement of this project should unravel novel mechanisms involving DNA methylation and intraganglionic oxidative metabolites on functional regulation of multiple pro-nociceptive genes, providing a mechanistic basis for how inflammation and stress engage sensory ganglia to induce prolonged persistent muscle pain. The anticipated outcomes should have broad translational implications for the development of therapeutic approaches targeting transcriptional machineries that regulate DNA methylation of a specific pro-nociceptive gene or a cluster of genes sharing similar epigenetic mechanisms.